We have been examining how sympathetic neurons chose the neurotransmitters that they will use and how target tissues acquire the appropriate complement of receptors and effector proteins. Descriptive studies from our laboratory have delineated a developmental change in neurotransmitter phenotype from noradrenergic to cholinergic in the sympathetic innervation of sweat glands in rodent footpads. Transplantation and culture experiments provide evidence that interactions with the traget tissue induce this change. Recent studies with an antiserum the recognizes the vesicular acetylcholine transporter (VAChT) suggested that the development of cholinergic function in sympathetic neurons, including those that innervate sweat glands, occurs prior to and does not require target contact. To clarify thse apparently contradictory findings, we directly compared the appearance of VAChT immunoreactivity in the sympathetic neurons that innervate sweat glands with the time that axons contact this target. We find that VAChT is not detectable in either the axons or cell bodies of sweat gland neurons until several days after target innervation. Before and during VAChT acquisition, the developing sweat gland innervation contains vesicular stores of catecholamines. An analysis of mutant mice that lack sweat glands was undertaken to determine whether VAChT expression requires target interactions and revealed that VAChT does not appear in the absence of glands. These findings, together with previous studies, confirm the target dependence of cholinergic function in the sympathetic neurons that innervate sweat glands. Previous studies revealed that the retrograde specification of neurotransmitter phenotype is mediated by a factor secreted by the target tissue. Similar changes are induced in cultured sympathetic neurons by sweat gland cells, or the neuropoietic cytokines leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), or cardiotrophin-1 (CT-1). None of these, however, is the sweat gland- derived differentiation activity. LIF, CNTF, and CT-1 act through the known receptors LIFR-beta and gp130 and well-defined signaling pathways including receptor phosphorylation and STAT3 activation. Therefore, to determine whether the gland-derived differentiation activity is a member of the LIF/CNTF cytokine family, we tested whether it acts via these same receptors and signal cascades. Blockade of LIFR-beta inhibited the sweat gland differentiation activity in neuron/gland cocultures, and extracts of gland-containing footpads stimulated tyrosine phosphorylation of LIFR-beta and gp130. Soluble footpad extracts induced the same changes in NBFL neuroblastoma cells as LIF and CNTF, including increased VIP mRNA, STAT3 dimerization and DNA binding, and stimulation of transcription from the VIP cytokine-responsive element. These findings indicate that the sweat gland-derived differentiation activity uses the same signaling pathway as the neuropoietic cytokines, and is likely to be a novel family member. Production of the transmitter differentiation factor by cultured sweat glands requires noradrenergic sympathetic innervation. To determine whether this is also true in vivo, we took advantage of transgenic mice in which the active site in the coding region of tyrosine hydroxylase was deleted. When, however, we examined sympathetic target tissues and the adrenal medulla of these mice, we discovered that there were significant stores of catecholamines present. One possible explanation for this surprising finding is that the transgenic animals were pigmented and tyrosinase in melanocytes, like tyrosine hydroxylase in neurons, can hydroxylate tyrosine to form DOPA. To determine whether tyrosinase was in fact the source of the catecholamines, we examined albino transgenic mice which lacked both tyrosinase and catalytic tyrosine hydroxylase. Catecholamines were not detectable in these animals. We are presently examining the neurotransmitter properties of the sweat gland innervation in the albino transgenic mouse to see if the alteration in transmitter properties occurs in the absence of catecholaminergic innervation.